Walder, J. a., et al., (1979), Complementary carrier peptide synthesis: General strategy and implications for prebiotic origin of peptide synthesis. Proc.Natl.Acad.Sci USA, vol. 76, pp. 51-55.
Ebata K., et al. (1995), Nucleic acids hyridization accompanied with excimer formation from two pyrene-labeled probes.
Photochemistry and Photobiology, vol. 62(5), pp. 836-839.
Nielsen P. E., (1995), DNA analogues with non phosphodiester backbones. Annu.Rev.Biophys. Biomol.Struct. vol.24, pp. 167-83.
Tam J. P., et al., (1995), Peptide synthesis using unprotected peptides through orthogonal coupling methods.
Proc.Natl.Acad.Sci. USA, vol.92, pp.12485-12489.
Uhlmann G. A. et al., (1990) Antisense Oligonucleotides: A New Therapeutic principle, Chemical Rev., vol. 90, pp.543-584.
Moser H. E. and Dervan P. B., (1987), Sequence-specific cleavage of double helical DNA by triple helix formation. Science, vol. 238, pp.645-650.
Tulchinsky E. et al., (1992) xe2x80x9cTranscriptional analsis of the mts 1 gene with the specific reference to 5xe2x80x2 flanking sequences. Proc.Natl.Acad.Sci USA, vol. 89, pp. 9146-9150.
The use of oligo(ribo)nucleotides and their analogues as anticancer and antiviruses theraupetic agents was first proposed several years ago. (Uhlmann, 1990) The great number of different modifications of the oligonucleotides and the methods of their use have since been developed.
Two basic interactions between oligonucleotides and nucleic acids are known (Moser and Dervan, 1987)
1. Watson-Crick base pairing (Duplex structure)
2. Hoogsten base pairing (Triplex structure)
Oligonucleotides can form duplex and/or triplex structures with DNA or RNA of cells and so regulate transcription or translation of genes.
It has been proposed that different substances which can cleave target nucleic acids or inhibit important cellular enzymes could be coupled to oligomers. The use of such conjugates as therapeutic agents has been described.(U.S. Pat. Nos., 5,177,198; 5,652,350).
Other methods are based on the coupling of different biologically active substances, such as toxins, to monoclonal antibodies which can then recognise receptors or other structures of cancer cells, or cells infected with viruses. Monoclonal antibodies can then specifically recognise cancer cells and in this way transport toxins to these cells. But these methods are inefficient due to the high level of nonspecific interactions between antibodies and other cells, which leads to delivary of the toxins or other biologically active compounds to the wrong cells.
In 1979 I. M. Klotz and co-authors proposed a method for complementary carrier peptide synthesis based on a template-directlyed scheme (J. A. Walder et al. 1979) The method proposed the synthesis of peptides on a solid support using unprotected amino acids, and the subsequent hybridization of oligonucleotides on the template. This method was established only for synthesis of peptides in vitro using solid supports of a different origin, and involved many synthesis steps to obtain peptides of the determined structure.
M. Masuko and co-authors proposed another method for in vitro detection of specific nucleic acids by excimer formation from two pyrene-labeled probes (Ebata, K. et al. 1995).
My invention allows the synthesis of different BACs of determined structure directly in living organisms only in cells which have specific RNA or DNA sequences. In this way, BACs will be delivered only to those cells where specific nucleic acids are produced.
xe2x80x9cNulceomonomerxe2x80x9d
The term xe2x80x9cnucleomonomerxe2x80x9d means a xe2x80x9cBasexe2x80x9d chemically bound to xe2x80x9cSxe2x80x9d moieties. Nucleomonomers can include nucleotides and nucleosides such as thymine, cytosine, adenine, guanine, diaminopurine, xanthine, hypoxanthine, inosine and uracil. Nucleomonomers can bind each other to form oligomers which can be specifically hybridised to nucleic acids in a sequence and direction specific manner.
The xe2x80x9cSxe2x80x9d moieties used herein include D-ribose and 2xe2x80x2-deoxy-D-ribose. Sugar moieties can be modified so that hydroxyl groups are replaced with a heteroatom, aliphatic group, halogen, ethers, amines, mercapto, thioethers and other groups. The pentose moiety can be replaced by a cyclopentane ring, a hexose, a 6-member morpholino ring; it can be aminoacids analogues coupled to base, bicyclic riboacetal analogues, morpholino carbamates, alkanes, ethers, amines, amides, thioethers, formacetals, ketones, carbamates, ureas, hydroxylamines, sulfamates, sulfamides, sulfones, glycinyl amides other analogues which can replace sugar moieties. Oligomers obtained from the mononucleomers can form stabile duplex and triplex structures with nucleic acids. (Nielsen P. E. 1995, U.S. Pat. No. 5,594,121).
xe2x80x9cBasexe2x80x9d
xe2x80x9cBasexe2x80x9d (designated as xe2x80x9cBaxe2x80x9d) includes natural and modified. purines and pyrimidines such as thymine, cytosine, adenine, guanine, diaminopurine, xanthine, hypoxanthine, inosine, uracil, 2-aminopyridine, 4,4-ethanocytosine, 5-methylcytosine, 5-methyluracil, 2-aminopyridine and 8-oxo-N(6)-methyladenine and their analogues. These may include, but are not limited to adding substituents such as xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH(3), xe2x80x94OCH(3), xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94NH(2), alkyl, groups and others. Also, heterocycles such as triazines are included.
xe2x80x9cNucleotidexe2x80x9d
Nucleotide as used herein means a base chemically bound to a sugar or sugar analogues having a phosphate group or phosphate analog.
xe2x80x9cOligomerxe2x80x9d
Oligomer means that at least two xe2x80x9cnucleomonomersxe2x80x9d (defined above) are chemically bound to each other. Oligomers can be oligodeoxyribonucleotides consisting of from 2 to 200 nucleotides, oligoribonucleotides consisting of from 2 to 200 nucleotides, or mixtures of oligodeoxyribonucleotides and oligoribonucleotides. The nucleomonomers can bind each other through phosphodiester groups, phosphorothioate, phosphorodithioate, alkylphosphonate, boranophosphates, acetals, phosphoroamidate, bicyclic riboacetal analogues morpholino carbamates, alkanes, ethers, amines, amides, thioethers, formacetals, ketones, carbamates, ureas, hydroxylamines, sulfamates, sulfamides, sulfones, glycinyl amides and other analogues which can replace phosphodiester moiety. Oligomers are composed of mononucleomers or nucleotides. Oligomers can form stable duplex structures via Watson-Crick base pairing with specific sequences of DNA, RNA, mRNA, rRNA and tRNA in vivo in the cells of living organisms or they can form stable triplex structures with double stranded DNA or dsRNA in vivo in the cells of living organisms.
xe2x80x9cAlkylxe2x80x9d
xe2x80x9cAlkylxe2x80x9d as used herein is a straight or branched saturated group having from 1 to 10 carbon atoms. Examples include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the like.
xe2x80x9cAlkenylxe2x80x9d
xe2x80x9cAlkenylxe2x80x9d as used herein is a straight- or branched-chain olefinically-unsaturated group having from two to 25 carbon atoms. The groups contain from one to three double bounds. Examples include vinyl (xe2x80x94CHdbdCH(2), 1-propenyl (xe2x80x94CHdbdCHxe2x80x94CH(3)), 2-methyl-1-propenyl (xe2x80x94CHdbdC(CH(3))xe2x80x94CH(3)) and the like
xe2x80x9cAlkynylxe2x80x9d
xe2x80x9cAlkynylxe2x80x9d as used herein is a straight or branched acetynically-unsaturated groups having from two to 25 carbon atoms. The groups contain from one to three triple bounds. Examples include 1-alkynyl groups include ethynyl (xe2x80x94CtbdCH), 1-propynyl (xe2x80x94CtbdCxe2x80x94CH(3)), 1-butynyl (xe2x80x94CtbdCxe2x80x94CH(2 xe2x80x94CH(3)), 3-methyl-butynyl (xe2x80x94CtbdCxe2x80x94CH(CH(3)) xe2x80x94CH(3)), 3,3-dimethyl-butynyl (xe2x80x94CtbdCxe2x80x94C(CH(3))(3)), 1-pentynyl (xe2x80x94CtbdCxe2x80x94CH(2, xe2x80x94CH(2 xe2x80x94CH(3)) and 1,3-pentadiynyl (xe2x80x94CtbdCxe2x80x94CtbdCxe2x80x94CH(3)) and the like.
xe2x80x9cArylxe2x80x9d
xe2x80x9cArylxe2x80x9d as used herein includes aromatic groups having from 4 to 10 carbon atoms. Examples include phenyl, naphtyl and like this.
xe2x80x9cHeteroalkylxe2x80x9d
xe2x80x9cHeteroalkylxe2x80x9d as used herein is an alkyl group in which 1 to 8 carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen) atoms. At any carbon atom there can be one to three substituents. The substituents are selected from: xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, xe2x80x94NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclic and like this groups.
xe2x80x9cHeteroalkenylxe2x80x9d
xe2x80x9cHeteroalkenylxe2x80x9d as used herein is an alkenyl group in which 1 to 8 carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen) atoms. At any carbon atom there can be one to three substituents. The substituents are selected from group xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclic and like this groups.
xe2x80x9cHeteroalkynylxe2x80x9d
xe2x80x9cHeteroalkynylxe2x80x9d as used herein is an alkynyl group in which 1 to 8 carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen) atoms. At any carbon atom there can be one to three substituents. The substituents are selected from group xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x2xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclic and like this groups.
xe2x80x9cHeteroarylxe2x80x9d
xe2x80x9cHeteroarylxe2x80x9d as used herein means an aromatic radicals comprising from 5 to 10 carbon atoms and additionally containing from and to three heteroatoms in the ring selected from group S, O or N. The examples include but not limited to: furyl, pyrrolyl, imidazolyl, pyridyl indolyl, quinolyl, benzyl and the like. One to three carbon atoms of aromatic group can have substituents selected from xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR, alkyl group. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclic or similar groups.
xe2x80x9cCycloheteroarylxe2x80x9d
xe2x80x9cCycloheteroarylxe2x80x9d as used herein means a group comprising from 5 to 25 carbon atoms from one to three aromatic groups which are combined via a carbocyclic or heterocyclic ring. An illustrative radical is fluorenylmethyl. One to two atoms in the ring of aromatic groups can be heteroatoms selected from N, O or S. Any carbon atom of the group can have substituents selected from xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR, alkyl group. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic and carbocyclic and like this groups.
xe2x80x9cCarbocyclicxe2x80x9d
xe2x80x9cCarbocyclicxe2x80x9d as used herein designates a saturated or unsaturated ring comprising from 4 to 8 ring carbon atoms. Carbocyclic rings or groups include cyclopentyl, cyclohexyl and phenyl groups. Any carbon atom of the group can have substituents selected from xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR, alkyl group. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic and carbocyclic and like this groups.
xe2x80x9cHeterocyclic Ringxe2x80x9d
xe2x80x9cHeterocyclic ringxe2x80x9d as used herein is a saturated or unsaturated ring comprising from 3 to 8 ring atoms. Ring atoms include C atoms and from one to three N, O or S atoms. Examples include pyrimidinyl, pyrrolinyl, pyridinyl and morpholinyl. At any ring carbon atom there can be substituents such as xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, halogen, xe2x80x94NH2, NO2, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94Oxe2x80x94S(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(O)(O)xe2x80x94Oxe2x80x94, xe2x80x94NHR, alkyl. Where R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic and carbocyclic and like this groups.
xe2x80x9cHybridizationxe2x80x9d
xe2x80x9cHybridizationxe2x80x9d as used herein means the formation of duplex or triplex structures between oligomers and ssRNA, ssDNA, dsRNA or dsDNA molecules. Duplex structures are based on Watson-Crick base pairing. Triplex structures are formed through Hoogsteen base interactions. Triplex structures can be parallel and antiparallel.
The word xe2x80x9chalogenxe2x80x9d means an atom selected from the group consisting of F (fluorine), Cl (clorine), Br (bromine) and I (iodine)
The word xe2x80x9chydroxylxe2x80x9d means an xe2x80x94OH group:
The word xe2x80x9ccarboxylxe2x80x9d means an xe2x80x94COOH function.
The word xe2x80x9cmercaptoxe2x80x9d means an xe2x80x94SH function.
The word xe2x80x9caminoxe2x80x9d meansxe2x80x94NH(2) or xe2x80x94NHR. Where R is alkyl, alkenyl, aryl, heteroaryl, heteroalkyl, alkynyl, heterocyclic, carbocyclic and like this groups.
xe2x80x9cBiologically Active Compounds (BACs)xe2x80x9d
xe2x80x9cBiologically active compound as defined herein include but are not limited to:
1) biologically active peptides and proteins consisting of natural aminoacids and their synthetic analogues L, D, or DL configuration at the alpha carbon atom selected from valine, leucine, alanine, glycine, tyrosine, tryptophan, tryptophan isoleucine, proline, histidine, lysin, glutamic acid, methionine, serine, cysteine, glutamine phenylalanine, methionine sulfoxide, threonine, arginine, aspartic acid, asparagin, phenylglycine, norleucine, norvaline, alpha-aminobutyric acid, O-methylserine, O-ethylserine, S-methylcysteine, S-benzylcysteine, S-ethylcysteine, 5,5,5-trifluoroleucine and hexafluoroleucine. Also included are other modifications of aminoacids which include but are not limited to, adding substituents at carbon atoms such as xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94NH2. The peptides can be also glycosylated and phosphorylated.
2) Cellular proteins which include but are not limited to: enzymes, DNA polymerases, RNA polymerases, esterases, lipases, proteases, kinases, transferases, transcription factors, transmembrane proteins, membrane proteins, cyclins, cytoplasmic proteins, nuclear proteins, toxins and like this.
3) Biologically active RNA such as mRNA, ssRNA, rsRNA and like this.
4) Biologically active alkaloids and their synthetic analogues with added substituents at carbon atoms such as xe2x80x94OH, xe2x80x94SH, xe2x80x94SCH3, xe2x80x94OCH3, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94NH2, alkyl straight and branched.
5) Natural and synthetic organic compounds which can be:
a) inhibitors and activators of the cellular metabolism;
b) cytolitical toxins;
c) neurotoxins;
d) cofactors for cellular enzymes;
e) toxins;
f) inhibitors of the cellular enzymes.
xe2x80x9cPrecursor(s) of Biologically Active Substances (PBAC(s))xe2x80x9d
xe2x80x9cPrecursors of biologically active compounds (PBACs)xe2x80x9d as used herein are biologically inactive precursors of BACs which can form whole BACs when bound to each other through chemical moiety(ies) xe2x80x9cmxe2x80x9d or simultaneously through chemical moieties xe2x80x9cmxe2x80x9d and xe2x80x9cm^1xe2x80x9d. xe2x80x9cmxe2x80x9d and xe2x80x9cm^1xe2x80x9d are selected independently from: xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, dbdNxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(S)Sxe2x80x94, xe2x80x94C(S)Oxe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94.
Biologically active peptides and proteins are synthesized from shorter biologically inactive peptides. These shorter peptides as used herein are also biologically inactive precursors of biologically active compounds.
Biologically active RNAs can be synthesized from biologically inactive oligoribonucleotides.
xe2x80x9cOligomer-PBACxe2x80x9d
xe2x80x9cOligomer-PBACxe2x80x9d as used herein means a precursor of a BAC (PBAC) which is chemically bound at the first and/or last mononucleomer at the 3xe2x80x2 and/or 5xe2x80x2 ends of the oligomer through the chemical moieties L^1 and/or L^2. Chemical moieties L^1 and L^2 can be bound directly to a base or to a sugar moiety or to sugar moiety analogues or to phosphates or to phosphate analogues,
xe2x80x9cOligomern-PAnxe2x80x9d
xe2x80x9cOligomern-PAnxe2x80x9d as used herein means the precursor of a biologically active protein or RNA which is chemically bound at the first and/or last mononucleomer at the 3xe2x80x2 and/or 5xe2x80x2 ends of the oligomer through the chemical moieties L^1 and/or L^2. n means the ordinal number of the oligomer of PA. PAs are biologically inactive peptides or biologically inactive oligoribonucleotides. Wherein n is selected from 2 to 300.
a) In Formulas 1 to 4 PBACs are designated as xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d
A-m-B is equal to a whole BAC xe2x80x9cTxe2x80x9d
xe2x80x9cmxe2x80x9d is selected independently from xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, dbdNxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(S)Sxe2x80x94, xe2x80x94C(S)O, xe2x80x94Nxe2x95x90Nxe2x80x94.
A-Oxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-NHxe2x80x94C(O)xe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)xe2x80x94NHxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)xe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-NHxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-dbdNxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)Oxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)Sxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(S)Sxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-Sxe2x80x94Sxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(S)Oxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-Nxe2x95x90Nxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
b) Biologically active compounds can be formed through moieties xe2x80x9cmxe2x80x9d and xe2x80x9cm^1xe2x80x9d. xe2x80x9cmxe2x80x9d and xe2x80x9cm^1xe2x80x9d are selected independently from: xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, dbdNxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(S)Sxe2x80x94, xe2x80x94C(S)O, xe2x80x94Nxe2x95x90Nxe2x80x94, so that 
is equal to biologically active compound xe2x80x9cTxe2x80x9d
a BAC is represented on figure
c) In Formulas 5 to 7, precursors of BACs (PBACs) are designated as xe2x80x9cPAnxe2x80x9d, where n is selected from 2 to 300. xe2x80x9cPAxe2x80x9d are peptides consisting of from 2 to 100 amino acids or oligoribonucleotides consisting of from 2 to 50 ribonucleotides.
{PA1-m-PA2-m-PA3-m- . . . -m-PAn-3-m-PAn-2-m-PAn-1-m-PAn} is equal to BAC. BACs in this case are proteins or RNAs. Proteins can be enzymes, transcription factors, ligands, signaling proteins, transmembrane proteins, cytolitical toxins, toxins, cytoplasmic proteins, nuclear proteins and the like.
This invention relates to the synthesis of biologically active compounds directly in the cells of living organisms. This is achieved by the hybridization of two or more oligomers to cellular RNA or DNA. These oligomers are bound to biologically inactive PBACs (oliogmer-PBACs) containing chemically active groups.
BAC can be synthesized only in those cells of living organisms which have specific RNA or DNA molecules of a determined sequence.
The principle Formulas of the invention are represented below: 
After hybridization of the xe2x80x9cOligomer-PBACsxe2x80x9d xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d to cellular RNA, DNA or dsDNA, the chemically active groups K^1 and K^2 of the oligomer-PBACs xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d interact with each other to form the chemical moiety xe2x80x9cmxe2x80x9d, which combines PBACs xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d into one active molecule of biologically active compound xe2x80x9cTxe2x80x9d. The degradation of the oligomers and/or linking moieties L^1 and L^2 by cellular enzymes or hydrolysis leads to the release of the synthesized BAC xe2x80x9cTxe2x80x9d directly into the targeted cells. After hybridization of the oligomer-PBACs to cellular RNA or DNA the distance between the 3xe2x80x2 or 5xe2x80x2 ends of the oligomer A and 5xe2x80x2 or 3xe2x80x2 ends of the oligomer B is from 0 to 7 nucleotides of cellular RNA, DNA or dsDNA. 
After hybridization of the xe2x80x9coligomer-PBACsxe2x80x9d xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d to cellular RNA, DNA or dsDNA the chemically active group K^2 of the oligomer-PBAC xe2x80x9cBxe2x80x9d interacts with the linking moiety L^1 of the oligomer-PBAC xe2x80x9cAxe2x80x9d to combine the PBACs through the chemical moiety xe2x80x9cmxe2x80x9d into one active molecule of biologically active compound xe2x80x9cTxe2x80x9d with the subsequent release of one PBAC xe2x80x9cBxe2x80x9d from the oligomer. The degradation of the oligomer and/or linking moieties L^1 by cellular enzymes or hydrolysis leads to the release of synthesized BAC xe2x80x9cTxe2x80x9d directly into the targeted cells. 
The chemically active group K^1 of the oligomer-PBAC A interacts with the linking moiety L^2 to combine the PBACs through the chemical moiety xe2x80x9cmxe2x80x9d into one active molecule of the biologically active compound xe2x80x9cTxe2x80x9d with the subsequent release of one PBAC xe2x80x9cBxe2x80x9d from oligomer xe2x80x9cBxe2x80x9d and the activation of the chemical moiety L^2. After activation, L^2 interacts with the linking moiety L^1 to release the biological compound xe2x80x9cTxe2x80x9d from the oligomer directly into targeted cells. 
After hybridization, of the xe2x80x9coligomer-PBACsxe2x80x9d xe2x80x9cAxe2x80x9d and xe2x80x9cBxe2x80x9d to cellular RNA, DNA or dsDNA, the chemically active group K^2 of the oligomer-PBAC xe2x80x9cBxe2x80x9d interacts with the linking moiety L^1 of the oligomer-PBAC xe2x80x9cAxe2x80x9d to combine the PBACs through the chemical moiety xe2x80x9cmxe2x80x9d. At the same time the chemically active group K^1 of the oligomer-PBAC xe2x80x9cAxe2x80x9d interacts with the linking moiety L^2 of the oligomer-PBAc xe2x80x9cBxe2x80x9d to form chemical moiety m^1. Which together with chemical moiety m combines two xe2x80x9cOligomer-PBACsxe2x80x9d into one active molecule of biologically active compound xe2x80x9cTxe2x80x9d, with the release of BAC from the oligomer. 
After simultaneous hybridization of xe2x80x9cOligomern-1-PAn-1xe2x80x9d and xe2x80x9cOligomern-PAnxe2x80x9d to cellular RNA or DNA, the chemically active groups K^1 and K^2 interact with each other to form the chemical moiety xe2x80x9cmxe2x80x9d between xe2x80x9cOligomern-1-PAn-1xe2x80x9d and xe2x80x9cOligomern-PAnxe2x80x9d correspondingly; This step is repeated in the cells n-1 times and combines n-1 times all xe2x80x9cPAnxe2x80x9ds into one active molecule of the biologically active compound xe2x80x9cPRxe2x80x9d which consists of n PAn so that compound {xe2x80x9cPAxe2x80x9d1-m-xe2x80x9cPAxe2x80x9d2-m-xe2x80x9cPAxe2x80x9d3-m-xe2x80x9cPAxe2x80x9d4-m- . . . -m-xe2x80x9cPAn-3xe2x80x9d-m-xe2x80x9cPAn-2xe2x80x9d-m-xe2x80x9cPAn-1xe2x80x9d-m-xe2x80x9cPAnxe2x80x9d} is biologically active compound xe2x80x9cPRxe2x80x9d. The degradation of the oligomers and/or linking moieties L^1 and L^2 leads to the release of the synthesized BAC xe2x80x9cPRxe2x80x9d directly into targeted cells of living organism. Here, n is selected from 2 to 2000; 
After simultaneous hybridization of xe2x80x9coligomern-1-PAn-1xe2x80x9d and xe2x80x9coligomern-PAnxe2x80x9d to cellular RNA, DNA or dsDNA, the chemically active group K^1 of xe2x80x9coligomern-PAnxe2x80x9d interacts with the linking moiety L^2 of xe2x80x9coligomern-1-PAn-1xe2x80x9d to bind PAn-1 and PAn through chemical moiety xe2x80x9cmxe2x80x9d. This step is repeated in the cells n-1 times and combines n-1 times all PAns after hybridization of all n xe2x80x9coligomer-PAnxe2x80x9ds into one active molecule of the biologically active compound xe2x80x9cPRxe2x80x9d, which consists of n PAs so that compound {PA1-m-PA2-m-PA3-m-PA4-m- . . . -m-PAn-3-m-PAn-2-m-PAn-1-m-PAn} is equal to the biologically active compound PR. The degradation of the oligomers and/or linking moieties L^1 by cellular enzymes or hydrolysis leads to the release of the synthesized BAC PR directly into targeted cells of living organism, here n is selected from to 2000; 
After simultaneous hybridization of xe2x80x9cOligomern-1-PAn-1xe2x80x9d and xe2x80x9coligomern-PAnxe2x80x9d to cellular RNA, DNA or dsDNA, the chemically active group K^1 of xe2x80x9coligomern-1-PAn-1xe2x80x9d interacts with the linking moiety L^2 of xe2x80x9coligomern-PAnxe2x80x9d to bind PAn-1 and PAn through chemical moiety xe2x80x9cmxe2x80x9d. After interaction of K^1 with L^2, L^2 is chemically activated so that it can interact with linking moiety L^1 of the oligomer-PAn-1, thus destroying the binding of the oligomern-1 to PAn-1. This process is repeated n-1 times, so that only whole BAC xe2x80x9cPRxe2x80x9d comprising from n PAns {PA1-m-PA2-m-PA3-m-PA4-m- . . . -m-PAn-3-m-PAn-2-m-PAn-1-m-PAn} is released directly into the targeted cells of living organisms, here n is selected from 2 to 2000.
The chemical moieties in the Formulas 1,2,3,4,5,6 and 7 are as follows:
m is selected independently from: xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94N(H)C(O)xe2x80x94, xe2x80x94C(O)N(H)xe2x80x94, xe2x80x94C(S)xe2x80x94Oxe2x80x94, xe2x80x94C(S)xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94C(O)xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Sxe2x80x94;
K^1 is selected independently from: xe2x80x94NH(2), dbdNH, xe2x80x94OH, xe2x80x94SH, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94R^1-C(X)xe2x80x94X^1-R^2;
K^2 is selected independently from: xe2x80x94NH(2), -dbd-NH, xe2x80x94OH, xe2x80x94SH, xe2x80x94R^1-C(X)xe2x80x94X^1-R^2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I;
L^1 is independently: chemical bond, xe2x80x94R^1-, xe2x80x94R^1-Oxe2x80x94Sxe2x80x94R-^2-, xe2x80x94R^1-Sxe2x80x94Oxe2x80x94R-^2-, xe2x80x94R^1-Sxe2x80x94Sxe2x80x94R^2-, xe2x80x94R^1Sxe2x80x94N(H)xe2x80x94R^2-, xe2x80x94R^1-N(H)xe2x80x94Sxe2x80x94R^2-, xe2x80x94R^1-Oxe2x80x94N(H)xe2x80x94R^2-, xe2x80x94R^1-N(H)xe2x80x94Oxe2x80x94R^2-, xe2x80x94R^1-C(X)xe2x80x94Xxe2x80x94R^2-;
L^2 is independently: chemical bond, xe2x80x94R^1, xe2x80x94R^1-Oxe2x80x94Sxe2x80x94R^2-, xe2x80x94R^1-Sxe2x80x94Oxe2x80x94R^2-, xe2x80x94R^1-Sxe2x80x94Sxe2x80x94R^2-, xe2x80x94R^1-Sxe2x80x94N(H)xe2x80x94R^2-, xe2x80x94R^1-N(H)xe2x80x94Sxe2x80x94R^2-, xe2x80x94R^1-Oxe2x80x94N(H)xe2x80x94R^2-, xe2x80x94R^1-N(H)xe2x80x94Oxe2x80x94R^2-, xe2x80x94R^1-C(X)xe2x80x94X^1-R^2-, xe2x80x94R^1-Xxe2x80x94C(X)xe2x80x94Xxe2x80x94C(X)xe2x80x94Xxe2x80x94R^2-;
R-^1 is independently: chemical bond, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl, carbocyclic, heterocyclic ring, X^1-P(X)(X)xe2x80x94X^1, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94X^1-S(X)(X)xe2x80x94X^1-, xe2x80x94C(O)xe2x80x94, xe2x80x94N(H)xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94X^1P(X)(X)xe2x80x94X^1-, xe2x80x94X^1-P(X)(X)xe2x80x94X^1-.
P(X)(X)xe2x80x94X^1, xe2x80x94X^1-P(X)(X)xe2x80x94X^1-P(X)(X)xe2x80x94X^1-P(X)(X)xe2x80x94X^1, xe2x80x94C(S)xe2x80x94, any suitable linking group;
R^2 is independently chemical bond, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl, carbocyclic, heterocyclic ring, X^1-P(X)(X)xe2x80x94X^1, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)(O)xe2x80x94, xe2x80x94X^1-S(X)(X)xe2x80x94X-^1-, xe2x80x94C(O)xe2x80x94, xe2x80x94N(H)xe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94X^1-P(X)(X)xe2x80x94X^1-, xe2x80x94X^1-P(X)(X)xe2x80x94X^1-P(X)(X)xe2x80x94X^1, xe2x80x94X^1-P(X)(X)xe2x80x94X^1-P(X)(X)xe2x80x94X^1-P(X)(X)xe2x80x94X^1, xe2x80x94C(S)xe2x80x94, any suitable linking group;
X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl;
X^1 is independently S, O, NH, Se, alkyl, alkenyl, alkynyl.
In Formulas 1,2,3,4,5,6 and 7 the linking moieties L^1 and L^2 are bound to the first and/or last mononucleomers of the oligomers at their sugar or phosphate moiety, or directly to base, or to sugar moiety analogues, or to phosphate moiety analogues, or to base analogues.
All the described schemes demonstrate that BACs can not be synthesized in non-targeted cells because the molar concentration of the chemically active groups is too low, and without hybridization of the oligomer-PBACs to the template, specific reactions can not occur. After hybridization of the oligomer-PBACs to a specific template, the concentration of the chemically active groups is sufficient for the chemical reaction between the chemical groups of PBACs to occur. The reaction leads to chemical bond formation between PBACs and subsequent formation of a whole BAC. The degradation of the oligomers and/or linking moieties of the oligomers with PBACs leads to the release of BACS directly into targeted cells. To synthesise directly in cellsbiologically active polymers such as proteins and RNAs of determined structure more than two PBACs are used. PBACs for synthesis of proteins or RNAs are designated as PAn. PAn are peptides or oligoribonucleotides. The mechanisms of the interaction of such PBACs are the same as in the synthesis of small biologically active compounds. The difference is that the PBACs (with the exception of the first and last PBACs) are bound simultaneously to the 5xe2x80x2 and 3xe2x80x2 ends of the oligomers so that the direction of synthesis of the biologically active protein or RNA can be determined.
Possible functions of BACs synthesized by proposed methods are: 1) Killing of cells, 2) Stimulation of the metabolism of cells 3) Blocking of important ion channels such as Na+, K+, Ca++ and other ion channels, in order to inhibit signal transmissions. BACs can be proteins, peptides, alkaloids, synthetic organic compounds. They can be cleaved into two or more precursors called PBACs. After interaction between the chemical groups of PBACs, whole BAC is formed through the moiety xe2x80x9cmxe2x80x9d.
a) In Formula 1,2,3 and 4 PBACs are designated as xe2x80x9cAxe2x80x9d and
A-m-B is equal to a whole BAC xe2x80x9cTxe2x80x9d
xe2x80x9cmxe2x80x9d is selected independently from xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, dbdNxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(S)Sxe2x80x94, xe2x80x94C(S)Oxe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94.
A-Oxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-NHxe2x80x94C(O)xe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)xe2x80x94NHxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)xe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-NHxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-dbdNxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)Oxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(O)Sxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(S)Sxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-Sxe2x80x94Sxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-C(S)Oxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
A-Nxe2x95x90Nxe2x80x94B is equal to a whole BAC xe2x80x9cTxe2x80x9d
b) A biologically active compound can be formed through the moieties xe2x80x9cmxe2x80x9d and xe2x80x9cm^1xe2x80x9d. xe2x80x9cmxe2x80x9d and xe2x80x9cm^1xe2x80x9d are selected independently from: xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94C(O)xe2x80x94, xe2x80x94C(O)xe2x80x94NHxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, dbdNxe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94C(O)Sxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94C(S)Sxe2x80x94, xe2x80x94C(S)Oxe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, so that 
is equal to biologically active compound xe2x80x9cTxe2x80x9d
This kind of interaction is represented in FIG. 4.
c) In Formulas 5, 6 and 7, precursors of BACs (PBACS) are designated as xe2x80x9cPAnxe2x80x9d, where n is selected from 2 to 2000. xe2x80x9cPAxe2x80x9d are peptides or oligoribonucleotides consisting of from 2 to 100 amino acids. n is the ordinal number of PA in a series of PAs and designates the sequence of binding of PAs to each other.
{xe2x80x9cPA1xe2x80x9d-m-xe2x80x9cPA2xe2x80x9d-m-xe2x80x9cPA3xe2x80x9d-m- . . . -m-xe2x80x9cPAn-3xe2x80x9d-m-xe2x80x9cPAn-2xe2x80x9d-m-xe2x80x9cPAn-1xe2x80x9d-m-xe2x80x9cPAnxe2x80x9d} is equal to BAC xe2x80x9cPRxe2x80x9d. BACs xe2x80x9cPRxe2x80x9d in this case are proteins or RNAs. Proteins can be cellular proteins, enzymes, transcription factors, ligands, signalling proteins, transmembrane proteins, cytolitical toxins, cytoplasmic and nuclear proteins and the like. RNAs are selected from mRNA, rsRNA and the like.